During early development, differentiating cells must activate genes previously not expressed that are required for cell type specification and organogenesis. For these genes to be turned on, chromatin must be de-compacted to allow transcription factors access to the DNA template. The goal of this research is to study the underlying mechanisms involved in chromatin remodeling. Specifically, we will examine chromatin opening at the albumin enhancer by the liver enriched transcription factor, HNF3, in a model of liver development. HNF3 is a pioneer transcription factor that can bind to and open compacted chromatin at the albumin enhancer in vitro in the absence of chromatin remodeling complexes. In order to establish the physiological relevance of this phenomenon, we plan to examine the role of HNF3 in chromatin remodeling in vivo using the F9 teratocarcinoma cell line. These cells, in the presence of retinoic acid, differentiate into visceral endoderm and turn on albumin gene expression as they differentiate. Because we are interested in examining the activation of the albumin enhancer, these cells will serve as a good model to ask questions about the role of HNF3 in chromatin opening. In order to determine changes in chromatin opening, we will use DNase I, micrococcal nuclease (MNase) and chromatin immunoprecipitation (ChIP) assays and compare chromatin accessibility to albumin gene upregulation. By mutating HNF3 binding sites on the albumin enhancer, we can determine whether HNF3 binding is required for chromatin remodeling at the albumin enhancer in vivo. Additional experiments with short hairpin RNAs (shRNAs) against HNF3 will determine whether HNF3 transcriptional activity is required to open chromatin at the albumin enhancer. In addition to determining the role of HNF3 in albumin enhancer remodeling, we will also examine a potential role for HNF3 in the activation of other liver specific genes, including transthyretin and alpha-fetoprotein. By fully understanding the mechanisms involved in gene activation during normal development, we can hopefully better understand aberrations in this process that may lead to developmental defects and cancer.